The present invention relates to veto cell preparations, methods of their manufacture and transplantation method using same which can be used to prevent or ameliorate immune rejection of donor organs, tissues or cells without inducing graft versus host disease (GVHD). More particularly, the present invention relates to a cell preparation for use in transplantation which preparation includes cells which are functional as veto cells but which are substantially devoid of alloreactive cells.
Transplantation of allogeneic and xenogeneic organs, tissues and cells is commonly practiced in humans in order to alleviate numerous disorders and diseases.
For example, bone marrow (BM) transplantation is increasingly used to treat a series of severe diseases in humans, such as for example, leukemia. However, bone marrow transplantation is limited by the availability of suitable donors, since transplanted tissues must traverse major histocompatibility barriers which can otherwise lead to graft rejection.
In view of such limitations, several approaches for enhancing graft acceptance have been suggested.
In one approach, cancer patients receiving autologous BM transplantation were treated with granulocyte colony-stimulating factor (G-CSF), resulting in mobilization of pluripotential stem cells from the marrow to the blood thereby increasing the number of cells which can be collected for autologous transplantation.
In another approach, major histocompatibility barriers in BM transplantation in leukemia patients were overcome by using a very large dose of stem cells, preferably a dose at least 3-fold greater than conventional doses used in T-cell-depleted BM transplantation, in particular a megadose of CD34+ hematopoietic progenitors (U.S. Pat. No. 5,806,529 to Reisner et al.).
Although the megadose approach facilitated permanent acceptance of allogeneic donor type skin grafts in mice 1, such an approach is not readily applicable for human transplantation since the number of stem cells which are required to attain this desirable goal may not be easily collected from human donors.
A difficult barrier for the engraftment of donor hematopoietic cell transplantation arises from the marked level of host hematopoietic and immune cells surviving mild preparatory regimens. Several studies have shown that this challenge can be successfully addressed in rodents by using large doses of bone marrow cells, adequately depleted of T-cells and utilized in conjunction with one form or another of tolerance inducing cells, also termed as veto cells.
Veto cell activity is defined as the capacity to specifically suppress cytotoxic T-cell precursors (CTL-p) directed against antigens of the veto cells themselves, but not against third party antigens 2 Several veto cells or bone marrow transplantation facilitating cells capable of suppressing cytotoxic T-cell precursors have been described3-11 
Interestingly, it has been shown that some of the most potent veto cells are of T-cell origin, and in particular a very strong veto activity was documented for CD8+ CTL lines or clones12-16.
The specificity of CTL veto cells was demonstrated by several studies to be unrelated to their T-cell receptor specificity17-19.
The suppression of effector CTL-p directed against the veto cells is both antigen-specific and MHC-restricted. This suppression results from the unidirectional recognition of the veto cell by the responding cytotoxic T-lymphocytes 18. Furthermore, it has been shown that this suppression is mediated by apoptosis18, 20.
Blocking experiments conducted with anti-CD8 or anti class I antibodies indicated that the elimination of host anti-donor CTL-p is induced via an interaction of CD8 molecules on the CTL veto cells with the a3 domain of class I molecules on the host CTL-p's 18. Support to this observation was provided by studies in which CD8 eDNA was introduced into clones lacking CD8 18. Further support was provided by experiments which demonstrated that CD8 molecules on the veto cells can directly induce apoptosis in effector cells 21. More recently, Asiedu et al. demonstrated that antibody mediated cross linking of CD8 on primate bone marrow veto cells, leads to an increased TGFβ production which induces apoptosis in the effector cells 22. Alternatively, it has been suggested that mouse bone marrow veto cells can induce apoptosis via Fas-Fas-L interaction 23.
The studies described hereinabove demonstrated that veto T-cell preparations can greatly facilitate graft tolerance in bone marrow transplantation. However, the veto T-cell preparations are inadequate for generating graft tolerance since such preparations still include a substantial amount of alloreacting donor T-cells which can lead to GVHD, thus limiting the successful implementation of this approach.
Reisner et al., 24, 28 describe the preparation of non-alloreactive anti-third party CTLs which can be used to enhance graft acceptance in mice. However, following the publication of this abstract and a more careful study, it was realized that the CTL preparation described therein is not depleted of T-cells capable of developing post transplantation into anti-host CTLs inflicting GVHD. Thus, this approach per se is not applicable for application in humans.
There is thus a widely recognized need for, and it would be highly advantageous to have, a novel veto cell preparation devoid of alloreactivity which can be used for substantially reducing rejection of transplanted organs, tissues or cells without generating GVHD, thereby leading to durable tolerance towards the transplanted, organs tissues or cells.